The broad long-term objectives of the currently funded proposal are to enhance our understanding of blood cell adhesion phenomena and to translate that knowledge into improved diagnosis, treatment, and prevention of disease. The first specific aim focuses on the structure, function, and activation mechanisms of the [unreadable]3 integrins, GPIIb/llla (allb[unreadable]3) and aV[unreadable]3, and the second specific aim focuses on the potential contribution of these receptors to the pathophysiology of sickle cell disease. One focus of the first aim is to identify the molecular mechanisms responsible for the processing of allb[unreadable]3 into its mature form. Our past studies of mutant integrin expression and our more recent studies of normal integrin biogenesis have been performed largely in HEK293T cells. Thus, a major limitation in these studies has been the absence of the megakaryocyte's protein trafficking and processing machinery. Our highest priority has been to validate our findings in human megakaryocytes and thus the availability of human embryonic stem cells (HESC) now makes this possible. We will take advantage of several recent advances in HESC and megakaryocyte biology: the availability of NIH-registered HESC lines, the availability of thrombopoietin, the recent advances in culturing murine embryonic cells that lead to the production of platelets, and advances in differentiating CD34+ human hematopoietic progenitors into megakaryocytes. The findings from the proposed studies have the potential to advance our understanding of the biogenesis of integrin receptors. Since these receptors play important roles in developmental biology, tumor growth and metastases, vascular biology and immunity, our data may have implications for normal physiology and disease states. Finally, since we will be trying to improve the in vitro production of platelets from human embryonic stem cell lines, it is possible that our findings will also have an impact on platelet transfusion therapy. The NIH-registered HESC line, ES03, will be purchased from ES Cell International. The long-term culture requirements for this cell line have been studied in great detail by Dr. Gordon Keller, who has agreed to train the key personnel in HESC culture techniques. Additionally, HESC culture protocols have been designed with the advice of Dr. Gordon Keller. [unreadable] [unreadable] [unreadable]